M. tuberculosis response to macrophage intracellular cues - Role of contact, phagosome pH, time, strain

A central feature of Mycobacterium tuberculosis (Mtb) pathogenesis is the ability of Mtb to survive within macrophages (MØ). Despite its critical importance our appreciation of the interplay between these two cells remains superficial. In this study we employed microarrays to conduct a stepwise dissection of Mtb-MØ interaction during the invasion of resting bone-marrow MØ. Contrary to many bacterial pathogens, engagement by MØ receptors without internalization did not alter Mtb gene expression. Subsequently, a high-resolution profile of Mtb invasion-linked gene expression was generated by assaying the Mtb transcriptome at 20 min intervals up to 2 hours post-infection. Transcriptional responses were detected within minutes of phagocytosis, including gene subsets with distinct temporal profiles. Pharmacological manipulation of phagosomal pH and in vitro acid stress studies revealed that vacuole acidification is an important trigger for differential gene expression. Finally, there are marked species-specific differences in the response of Mtb and M. bovis BCG to intraphagosomal cues. Keywords: time-course, species-specific expression, in vitro and in vivo acid stress M. tuberculosis CDC1551 or BCG RNA was isolated from macrophage associated bacteria using a guanidine thiocyanate based lysis/RNA stablization buffer that rapidly lyses macrophages while permeating mycobacteria to "freeze" and stabilize the transcriptome. Due to limited RNA yields from intracellular bacteria, total bacterial RNA was linearly amplified, incorporating amino-allyl UTP during in vitro transcription. aRNA were post-labeled with amine reactive Alexa dyes and samples were hybridized to spotted oligo arrays in treated vs control dual channel format. A minimum of two biological replicates was conducted for each assay. Contact-induced gene expression using cytochalasin D-treated macrophages and pH induced gene expression using concanamycin A treated macrophages (2 biological replicates), CDC1551 and BCG 2hr and 24hr intraphagosomal profiles (5 biological replicates), in vitro acid stress at pH 5.5 and 6.5 (3 biological and 2 technical replicates - 6 total arrays) for each condition.